Class II and class I proteins encoded by genes of the Major Histocompatability Complex (MHC) on chromosome 6 in humans play an essential role in regulating the immune system. MHC class II molecules, which are expressed in antigen-presenting cells such as macrophages, B cells, monocytes and some epithelial cells, form complexes with antigen peptides ("MHC class II antigen complexes") that are displayed on the surface of antigen-presenting cells for recognition by CD4+T lymphocytes (helper T cells). Helper T cell recognition results in release of lymphokines and T-dependent activation of B cells, which, in turn, lead to activation of macrophages and release of antibodies from B cells, leading to the killing or elimination of invading microorganisms. MHC class I molecules, which are expressed in virtually all nucleated cells, form complexes with antigen peptides ("MHC class I antigen complexes") that are displayed on the cell surface for recognition by CD8+cytotoxic T lymphocytes (CTLs). Presentation of an endogenous or "self" peptide by the MHC class I antigen complex is protective, the CTLs that would otherwise recognize the surface complex and attack the presenting cell (i.e., autoreactive CTLs) having been eliminated (deleted) from the immune system repertoire; and presentation of an exogenous (foreign or "non-self") peptide (or a mutated endogenous peptide) by the MHC class I antigen complex elicits CTL attack and cytolytic destruction of the infected or diseased cell.
The peptides that complex with MHC molecules are approximately eight to twenty-four amino acids in length. In the case of class II antigen complexes, the peptides are derived from partial proteolysis and processing of extracellular antigenic proteins incorporated by the cell through phagocytosis or pinocytosis or possibly surface processing. Thus, the immune recognition events mediated by MHC class II antigen complexes are a primary defense to invading microorganisms (e.g., bacteria, parasites) or foreign substances (e.g., haptens, transplant tissues) introduced to the cells of the immune system via the circulatory or lymph systems. In the case of class I antigen complexes, the antigen peptides are derived from intracellular processing of proteins. Thus, MHC class I antigen complexes either mark the cell as a normal endogenous cell, which elicits no immune response, or mark the cell as an infected cell (e.g., as in the case of a virus-infected cell exhibiting intracellularly processed viral peptide in the surface MHC class I complex) or a transformed cell (e.g., such as a malignant cell), which marks the cell for attack by CTLs.
Proper intracellular processing of antigen peptides for MHC class I complexing and presentation involves several steps. One of these steps is transport of the peptides from the cytosol into the endoplasmic reticulum (ER), where coupling of the antigen peptide with the MHC class I molecule takes place. The MHC class I antigen complex migrates to the cell surface for presentation and possible recognition by T cells. Unsuccessful transport of peptides into the ER, or other abnormalities leading to faulty class I antigen complex formation or presentation, can lead to a failure in recognizing autologous cells as "self". For example, defects in genes coding for transporter proteins have been discovered to be an underlying cause of several autoimmune diseases (Faustman et al., Science, 254:1756-1761 (1991); U.S. Pat. No. 5,538,854).
Transporter associated with Antigen Processing, or TAP, proteins transport peptide fragments of eight or more amino acids from the cytosol of a cell into the lumen of the ER, where the peptides are bound by MHC class I proteins to form an antigen complex, which ultimately is displayed on the surface of the cell (see, e.g., Powis et al., Immunogenetics, 37:373-380 (1990)). The TAP protein is a heterodimer of the products of the TAP1 and TAP2 genes, which are also located in the MHC region of the genome. Each subunit of the TAP1/TAP2 heterodimer forms an ATP-binding domain and a domain that criss-crosses the membrane six to eight times, and both subunits are required to form a peptide binding site and to translocate peptide into the ER (Androlewicz et al., Proc. Natl. Acad. Sci. USA, 91(26): 12716-12720 (1994); Hill et al., Proc. Natl. Acad. Sci. USA, 92: 341-343 (1995).)
The role of TAP in mediating the supply of antigen peptides transported into the ER and ultimately displayed by MHC class I molecules has caused close scrutiny of the range of peptides capable of translocation by TAP, to determine whether TAP is a further restrictive factor in immune diversity. (See, Hill et.al., ibid.; Howard., Proc. Natl. Acad. Sci. USA, 90: 3777-3779 (1993).) Whereas gene rearrangement during ontogeny of T cells generates an enormous variety of T cell receptor specificities, making the recognition capability of the immune system very diverse, there has not been discovered any corresponding mechanism for diversifying the presentation capability of an individual's immune system. Although small variations in MHC allotypes result in different repertoires of antigen peptides being complexed and presented by MHC molecules, lending diversity to antigen presentation across a species, an individual's MHC haplotypes restrict the range of antigens that can be effectively displayed. The specificity of the TAP transport mechanism also shapes the repertoire of antigen complexes presented to the immune system, in that only peptides capable of translocation by TAP are made available for complexing in the ER with MHC class I. (See, Howard, ibid.)
The peptide specificity of TAP proteins has been studied in three species thus far: human, mouse and rat. In the rat, it was shown that different alleles of the TAP2 gene gave rise to functional polymorphism, i.e., the different alleles transported sets of peptides that differed in C-terminal residues. (Powis et al., Immunity, 4(2):159-165 (1996); Powis et al., Nature, 357:211-215 (1992).) In the human and mouse, however, investigation of several polymorphs of TAP1 and TAP2 did not reveal any alteration in the spectrum of peptides transported, and it has been generally concluded that although in mice and humans the TAP1 and TAP2 proteins are genetically polymorphic, they are functionally monomorphic, the sequence alterations of the allotypes causing no shift in the types of peptides translocated by TAP. (Schumacher et al., Proc. Natl. Acad. Sci. USA, 91(26):13004-13008 (1994); Obst et al., Eur. J. Immunol., 25:2170-2176 (1995); Daniel et al., J. Immunol., 159:2350-2357 (1997).
It has now been discovered that the human TAP1 and TAP2 genes produce several splice variants that differ structurally and functionally from the known TAP1 and TAP2 proteins, and a functional TAP2 splice variant gene product, designated TAP2iso, has been characterized and its full coding sequence isolated. The TAP heterodimer including the TAP2iso splice variant, surprisingly, preferentially translocates a different set of peptides than the TAP heterodimer including TAP2. These discoveries have led to a revision described herein of the model of peptide transport into the lumen of the ER for MHC class I complexing; and a new level of diversity in the presentation of antigen complexes, akin in some respects to the diversity of T cell receptors in the recognition of such complexes, has been exposed for the first time.